Selective culture medium and method for detecting carbapenem-resistant bacteria in a test sample

ABSTRACT

The present invention relates to a culture medium comprising a carbapenem, a carbapenemase activator and a M-type penicillin. It also relates to a method for detecting carbapenem-resistant bacteria in a test sample using said culture medium.

FIELD OF THE INVENTION

The present invention relates to a selective culture medium and a method for detecting carbapenem-resistant bacteria, in particular carbapenemase producers, in a test sample.

BACKGROUND OF THE INVENTION

Carbapenemase-producing bacteria isolates are increasingly identified throughout the world (1-8). Their early detection is becoming a major issue in the field of clinical microbiology in order to prevent their spread and preserve the efficacy of carbapenems which are becoming the antibiotics of last resort for treating severe infections. Moreover, carbapenemases are usually associated to many other non-beta-lactam resistant determinants giving rise to multidrug and pandrug resistance. Therefore, due to current population exchange and travel, early recognition of carbapenemase producers is becoming mandatory whatever the antibiotic policy or rate of multidrug-resistant nosocomial infections.

The vast majority of acquired carbapenemases belong to three of the four known classes of beta-lactamases, namely Ambler class A, Ambler class B (metallo-beta-lactamases (MBLs)) and Ambler class D (oxacillinases (OXAs)). These three classes of carbapenemases confer clinical resistance to carbapenems. Consequently, carbapenemase-producing bacteria isolates from these three classes have been involved in nosocomial outbreaks (1,2,7,9,10).

The spread of these three distinct classes of carbapenemases varies significantly worldwide. For example, KPC producers (Ambler class A) are identified mostly in the Americas and Southern Europe, while IMP, VIM, NDM-1 (Ambler class B) are extensively identified worldwide with a main reservoir for NDM-1 in the Indian subcontinent. As for OXA-48-like enzymes (Ambler class D), it was first identified in Klebsiella pneumoniae in Turkey in 2003, where it is highly prevalent (11). However, OXA-48-producing bacteria, in particular Enterobacteriaceae, have also been reported in several other countries such as France, Belgium, United Kingdom, Germany Egypt, India and North Africa suggesting its widespread nature (1,6,12).

As previously mentioned, early recognition of carbapenemase producers is critical to prevent their spread. However, the level of reduced susceptibility to carbapenems may vary significantly among carbapenemase producers making their detection difficult (11,12,13,14,15).

In particular, although OXA-48-like enzymes remain susceptible to extended-spectrum cephalosporins, they confer resistance to penicillins and reduced susceptibility to carbapenems, thereby making the clinical laboratory detection of OXA-48-like producing isolates difficult. In fact, it has been identified in multidrug-resistant isolates, which often accumulate multiple resistance mechanisms, including production of extended-spectrum beta-lactamases (ESBLs). Most carbapenemase producers co-express ESBLs, but several OXA-48-like producing isolates that do not carry ESBL genes may remain susceptible to extended-spectrum cephalosporins (11).

Commercially available media for detecting carbapenemase producers exist. Examples of such media are the carbapenem-containing CHROMagar KPC (CHROMagar company, Paris, France) and the cefpodoxime-containing chromogenic ChromID ESBL medium (bioMerieux, La Balme-les-Grottes, France) which is designed to screen for ESBL producers (16-17). In addition, each medium contains a chromogenic molecule which may contribute to species recognition. The main disadvantage of screening carbapenemase producers using the ChromID ESBL medium is that it cannot detect OXA-48-like producers which are susceptible to cefpodoxime in the absence of co-production of an ESBL (12). In addition, this medium may lack specificity since non-carbapenemase ESBL producers are co-selected on that medium. As for the CHROMagar KPC medium, its main disadvantage is its lack of sensitivity since carbapenemase producers with low level of resistance to carbapenems are not efficiently detected on this medium (12).

Finally, WO 2010/010083 discusses a method for direct detection and differentiation of carbapenem-resistant bacteria in a sample comprising (i) inoculation with said sample of a culture medium comprising at least meropenem and/or ertapenem and at least one chromogenic agent, (ii) incubation of said culture medium under conditions allowing the growth of carbapenem-resistant bacteria, and (iii) detection of colonies formed on said culture medium corresponding to carbapenem-resistant bacteria. The application also discloses a culture medium suitable for use in such a method. However, the culture medium used in this method is neither sensitive enough, nor specific enough to detect carbapenemases producers with reduced susceptibility to carbapenems.

SUMMARY OF THE INVENTION

To facilitate the detection of carbapenem-resistant bacteria in the field of clinical microbiology, and in particular, carbapenemases producers with reduced susceptibility to carbapenems, the Applicant has developed a new reliable and highly sensitive culture medium comprising a combination of a carbapenem, a carbapenemase activator and a M-type penicillin. The advantage of this new medium for the detection of carbapenemase producers is that it is more sensitive and specific than existing media. Furthermore, neither a high level of Minimum Inhibitory Concentrations (MICs) of carbapenems (which is required in the CHROMagar KPC medium) nor co-expression of an Enlarged Spectrum Beta-Lactamases (ESBL) (which is required in the ChromoID ESBL media) is needed. Moreover this medium can be useful for the detection of carbapenemase producers in human faeces which also contain a large amount of ESBL producers and/or a large amount of AmpC cephalosporinases, the latter activity being inhibited by M-type penicillins. This last point could be more relevant in countries with high rates of these types of isolates.

Therefore, the present invention relates to a culture medium comprising a carbapenem, a carbapenemase activator and a M-type penicillin.

The present invention also relates to a method for detecting carbapenem-resistant bacteria in a test sample comprising the following successive steps:

-   -   a) inoculating a culture medium as defined herein with said test         sample,     -   b) incubating said culture medium under conditions suitable for         growth of carbapenem-resistant bacteria,     -   c) detecting colonies formed on said culture medium         corresponding to carbapenem-resistant bacteria.

DETAILED DESCRIPTION OF THE INVENTION

Definitions:

As used herein, “test sample” means any liquid or solid material to be tested which may contain carbapenemase-producing bacteria. The preferred test sample is a biological sample.

As used herein, “biological sample” means any biological sample derived from a subject. Examples of such samples include fluids, skin swabs, tissues, cell samples, etc. Preferred biological samples are saliva, whole blood, serum, plasma, urine or faeces.

As used herein, “subject” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably a subject according to the invention is a human.

Culture Medium:

As previously mentioned, early recognition of carbapenemase producers is critical to prevent their spread. However, a difficulty remains in that the level of reduced susceptibility to carbapenems may vary significantly among carbapenemase producers making their detection difficult.

As a solution to this problem, the Applicant has developed a new culture medium which is reliable, highly sensitive and more specific than existing media for the detection of carbapenemase producers.

The present invention therefore relates to a culture medium comprising a carbapenem, an activator of specific types of carbapenemases and a M-type penicillin.

Typically the concentration of carbapenem is comprised between 0.005 to 4 μg/ml. For example, the concentration of carbapenem can be comprised between 0.01, 0.02, 0.05, or 0.1 to 0.1, 0.2, 0.5 or 1 μg/ml, for example between 0.2 and 0.5 μg/ml. For example the carbapenem concentration is about 0.25 μg/ml.

The preferred concentration will typically depend on the carbapenem used.

For ertapenem, the preferred concentration is comprised between 0.1 to 1 μg/ml, preferably between 0.2 and 0.5 μg/ml and more preferably the ertapenem concentration is about 0.25 μg/ml.

For doripenem, the preferred concentration is comprised between 0.01, to 0.1 μg/ml, preferably between 0.015 and 0.05 μg/ml and more preferably the doripenem concentration is about 0.025 μg/ml.

A low concentration of carbapenem will enable the selection of carbapenemases producers with reduced susceptibility to carbapenems, the carbapenemase activator will increase the expression of Ambler Class B producers, while the M-type penicillin will prevent the growth of isolates which are resistant to carbapenems by production of cephalosporinases (and not carbapenemases) (+/−associated to outer membrane permeability defects), thereby reinforcing the selectivity of the medium. Such cephalosporinases are either of type AmpC or with broadened substrate activity, and can be found in species such as Enterobacter cloacae, Enterobacter aerogenes, Serratia marcescens (i.e. Ambler Class C producers).

According to the invention, the carbapenem is selected from the group consisting of biapenem, ertapenem, doripenem, imipenem, meropenem, tebipenem, panipenem and mixtures thereof. Preferably the carbapenem is ertapenem.

Based on molecular studies, carbapenemases can be further divided into two types: serine enzymes possessing a serine moiety at the active site (Ambler Class A, C, and D), and MBLs (Ambler Class B) which require divalent cations, usually zinc, as metal cofactors for enzyme activity, thereby facilitating hydrolysis of the bicyclic beta-lactam ring (18).

Thus, according to the invention, in order to increase the expression of Ambler Class B carbapenemase producers, the culture medium comprises a carbapenemase activator, which is selected from the group consisting of divalent cations and mixtures thereof.

Typically, in the case of carbapenemases of Ambler class B, the activator is a divalent cation or salt thereof selected from the group consisting of manganese, cobalt, nickel, cadmium, mercury, zinc and mixtures thereof (see Biochem J. 1974; 143(1):129-35 and the Journal of Biological Chemistry vol. 285, NO. 7, 4570-4577). Preferably, the carbapenemase activator is zinc.

Typically, the concentration of carbapenemase activator which is found in the culture medium is comprised between 10 or 30 and 100 μg/ml, preferably between 50 and 80 μg/ml, and more preferably the concentration of carbapenemase activator is about 70 μg/ml.

According to the invention, the M-type penicillin (also called methicillin type penicillin) is selected from the group consisting of cloxacillin, dicloxacillin, flucloxacillin, oxacillin, methicillin, nafcillin and mixtures thereof. Preferably the M-type penicillin is cloxacillin.

Typically, the concentration of M-type penicillin which is found in the culture medium is comprised between 100 and 500 μg/ml, preferably between 200 and 300 μg/ml, and more preferably the concentration of M-type penicillin is about 250 μg/ml.

Although, the culture medium according to the invention preferably comprises ertapenem, zinc and cloxacillin, any combination of carbapenem, carbapenemase activator and M-type penicillin as disclosed herein may be used in the culture medium of the present invention.

Typically, the culture medium according to the invention is an agar culture medium, wherein the culture medium is, for example, agar-based. Alternatively, the culture medium may be a broth liquid medium.

In addition, to the combination of carbapenem, carbapenemase activator and M-type penicillin, the culture medium according to the invention also comprises nutrients necessary to support the growth, proliferation and survival of the bacteria. Thus, an appropriate culture medium according to the invention typically comprises, in addition to the combination of carbapenem, carbapenemase activator and M-type penicillin, a minimal medium in which bacteria can grow. Typical examples of such minimal media are Drigalski, Tryticase soy and MacConckey.

Preferably the medium is selective for Gram-negative bacteria such as the Drigalski medium. Alternatively, selection of Gram negative bacteria may be based on antibiotic-antifungal drug association using a non-selective media such as Trypticase soy agar. In that case, the medium can comprise an antibiotic specific for gram positive bacteria such as a glycopeptide antibiotic or a lipopeptide antibiotic, such as daptomycin. For example the antibiotic can be a glycopeptide antibiotic selected from the group consisting of vancomycin, teicoplanin, telavancin, ramoplanin, and decaplanin. Typically the medium can comprise an antifungal drug, such as a polyene antifungal drug, for example amphotericin B.

Typically, the culture according to the invention may comprise a chromogenic component. A chromogenic component may facilitate species recognition. Suitable chromogenic components are well known and are used for example in the KPC Chromagar or ChromID ESBL or CRE screening media

Method of Detection:

According to another aspect of the invention, there is provided a method for detecting carbapenem-resistant bacteria in a test sample comprising the following successive steps:

-   -   a) inoculating a culture medium as defined herein with said test         sample,     -   b) incubating said culture medium under conditions suitable for         growth of carbapenem-resistant bacteria,     -   c) detecting colonies formed on said culture medium         corresponding to carbapenem-resistant bacteria.

Typically, once colonies have been detected on the culture medium, further techniques may be used to characterize the beta-lactamase content. Currently, there exist various methods for detecting carbapenemase producers. First, phenotypic-based techniques for in vivo production of carbapenemase such as the “Etest®” and the “Hodge-Test” can be used. Alternatively, molecular detection techniques for carbapenemase genes may be used. Finally, beta-lactamase identification using chromogenic molecules, biochemical, iodometric tests and acidimetric tests can be used as well as molecular techniques for identification of corresponding genes.

According to the present invention, the method may be used to detect carbapenem-resistant bacteria, in particular carbapenemase-producing bacteria, involved in nosocomial and community-acquired infections.

Typically, the carbapenem-resistant bacteria and in particular the carbapenemase-producing bacteria which are detected by the method of the invention have a reduced susceptibility to carbapenems. As used herein, “reduced susceptibility” means decreased susceptibility as compared to wild-type carbapenem susceptibility.

Typically, the carbapenem-resistant bacteria are bacteria, such as carbapenemase-producing bacteria, containing a gene coding for a beta-lactamase of the Ambler class A, B and/or D.

Preferably, the carbapenem-resistant bacteria are selected from the Enterobacteriaceae family and more preferably from the genera consisting of Citrobacter, Enterobacter, Escherichia, Klebsiella and Providencia.

In an embodiment of the invention, the method disclosed herein can be applied directly to a raw test sample comprising a mixture of bacteria without having to isolate the various bacterial strains present in the test sample.

The incubation conditions allowing the growth of carbapenem-resistant bacteria are well-known to the person skilled in the art and are not different from the usual methods.

Since ertapenem shows very good or good stability in an agar medium (2 to 4 weeks), a detection method like the one of the present invention can be carried out effectively over several months.

The invention will further be illustrated in view of the following examples.

EXAMPLES Example 1 Evaluation of the ETP-Cloxa-Zinc Agar Culture Medium

Applicant evaluated an in-house prepared ertapenem-cloxacillin-zinc (hereinafter, referred to as SUPERCARBA medium) containing agar as an alternative to CHROMagar KPC and ChromID ESBL.

Ertapenem was added at a low concentration of 0.25 μg/ml, ZnSO₄ (70 μg/ml) was added to improve growth of metallo-beta-lactamase (MBL)-producers (3) and cloxacillin (250 μg/ml) was used to prevent growth of isolates expressing cephalosporinases with broadened substrate activity, such as Enterobacter cloacae, Enterobacter aerogenes, and Serratia marcescens.

Forty-seven carbapenemase-producing isolates belonging to various enterobacterial species of worldwide origin were included in the study. These strains had been previously characterized for beta-lactamase content at the molecular level. The strains were as follows:

-   -   KPC-producers (n=4),     -   VIM/IMP-producers (n=5),     -   NDM-1-producers (n=16),     -   OXA-48 producers (n=19),     -   OXA-181-producers (n=3).         Twenty-nine out of forty-five carbapenemase producers         co-expressed an ESBL (Table 1).

Strains that did not express any carbapenemase were used as control. These control strains were:

-   -   ertapenem susceptible isolates producing an ESBL (n=6,         identified as “b” in Table 1),     -   ertapenem susceptible isolates producing a high-level AmpC (n=5,         identified as “c” in Table 1),     -   isolates with reduced susceptibility to ertapenem due to         over-expressed AmpC (n=4, identified as “d” in Table 1),     -   isolates with reduced susceptibility to ertapenem due to ESBL         and/or porin deficiency (n=12, identified as “e” in Table 1).

Using an inoculum of ˜2×10⁷ CFU/ml (range, 1.5×10⁷ to 3.5×10⁸ CFU/ml), serial 10-fold dilutions of the isolates were made in normal saline and 100 μl were plated onto SUPERCARBA containing Drigalski agar. Viable bacteria were counted after 24 hours of culture at 37° C. and growth on selective SUPERCARBA media was compared to growth on Drigalski agar without SUPERCARBA.

The lowest limit of detection of VIM and IMP producers ranged from 1×10¹ to 1×10⁶ CFU/ml (Table 1). Although addition of zinc sulfate helped to lower this detection limit for VIM-producers, IMP-producing isolates which were not always efficiently detected on this medium.

The lowest limit of detection of OXA-48, OXA-181, NDM-1 and KPC producers was 1×10¹ to 1×10² CFU/ml (Table 1). This result is, to the Applicant's knowledge, one of the most efficient ways to detect carbapenemase-producing isolates. Only one isolate (NDM-1 producing P. stuartii isolate) was not efficiently detected on this SUPERCARBA medium (Table 1). Low level of detection of this isolate might be explained by the low MIC of ertapenem (0.38 μg/ml) and weak expression of NDM-1.

As expected, OXA-181-producing K. pneumoniae were efficiently detected on SUPERCARBA. Surprisingly however, a very weak detection was obtained for the OXA-181-producing Providencia rettgeri despite high MICs of carbapenems (Table 1). With respect to P. rettgeri, contribution of AmpC cephalosporinases and impermeability to many β-lactam molecules may be very important compared to that of carbapenemases for providing the in vivo observer level of carbapenem resistance.

As expected, isolates that did not express any carbapenemase (i.e. AmpC and/or ESBL positive isolates) were inhibited on SUPERCARBA (with a detection limit of >10⁷ CFU/ml). In particular, cloxacillin helped to prevent growth of isolates expressing cephalosporinases with broadened substrate activity (Table 1). Nevertheless, among 12 ertapenem non-susceptible isolates (one C. freundii and eleven K. pneumoniae isolates) for which porin loss was very likely involved in ertapenem resistance, 50% (n=6) were detected on SUPERCARBA (lower detection limit <10² CFU/ml) (Table 1, species identified with“e”).

Addition of zinc sulfate was useful for eliminating part (up to 42%, n=5) of non-carbapenemase producing ertapenem-resistant K. pneumoniae isolates (19, 20, 21). Further studies are needed to investigate the effect of zinc on the outer-membrane protein profile of K. pneumoniae.

TABLE 1 Sensitivity of detection of SUPERCARBA medium for 45 carbapenemase- and/or ESBL/AmpC-producing enterobacterial isolates. Lowest Detection MIC (μg/ml) of drug^(a): limit Strains Beta-lactamases IPM ETP MEM (CFU/ml) OXA-48 K. pneumoniae BIC OXA-48 0.5 2 0.5 1 × 10¹ K. pneumoniae CHA OXA-48 + TEM-1 0.38 1 0.5 1 × 10¹ K. pneumoniae EGY OXA-48 + CTX-M-15 2 3 2 1 × 10¹ K. pneumoniae BEL OXA-48 1 4 1 1 × 10¹ K. pneumoniae RAM OXA-48 1 4 1 1 × 10¹ K. pneumoniae LIB OXA-48 >32 >32 >32 1 × 10¹ K. pneumoniae BEY OXA-48 + CTX-M-15 + TEM-1 0.38 0.38 0.38 1 × 10¹-1 × 10² K. pneumoniae DAL OXA-48 + CTX-M-15 + TEM-1 0.38 2 0.38 1 × 10¹ K. pneumoniae ROB OXA-48 + CTX-M-15 + TEM-1 0.38 3 0.5 1 × 10¹ K. pneumoniae SCO OXA-48 0.5 0.75 0.25 1 × 10¹ E. cloacae TUR OXA-48 + SHV-5 0.5 0.5 0.5 1 × 10¹ E. coli BER OXA-48 + CTX-M-15 0.38 1.5 0.19 1 × 10¹ E. coli AME OXA-48 + CTX-M-24 0.25 0.5 0.19 1 × 10¹ E. coli GOG OXA-48 + CTX-M-24 0.5 1.5 0.25 1 × 10¹ E. coli NAA OXA-48 + CTX-M-24 0.5 2 0.25 1 × 10¹ E. coli HAN OXA-48 + CTX-M-15 3 16 1 1 × 10¹-1 × 10² E. coli BOU OXA-48 + CTX-M-15 0.5 0.75 0.125 1 × 10¹-1 × 10² E. coli BON OXA-48 + CTX-M-24 + TEM-1 0.38 0.5 0.19 1 × 10² E. coli BOK OXA-48 + CTX-M-15 0.25 0.38 0.19 1 × 10² OXA-181 K. pneumoniae OMA OXA-181 + CTX-M-15 + OXA-1 0.5 2 0.5 1 × 10¹ K. pneumoniae HOL OXA-181 + CTX-M-15 1 4 1 1 × 10¹ P. rettgeri RAP OXA-181 + OXA-1 8 1 2 5 × 10² NDM-1 K. pneumoniae UK NDM-1 + CTX-M-15 + CMY-4 + >32 >32 >32 1 × 10¹ OXA-1 K. pneumoniae 6642 NDM-1 + CTX-M-15 + OXA-1 + 1 16 3 1 × 10¹ GEN OXA-10 K. pneumoniae 6759 NDM-1 + CTX-M-15 + OXA-1 + 12 >32 >32 1 × 10¹ GEN OXA-9 + OXA-10 + CMY16 K. pneumoniae 1 OMA NDM-1 + CTX-M-15 + OXA-1 + >32 >32 >32 1 × 10¹ OXA-9 K. pneumoniae 2 OLA NDM-1 + OXA-1 1.5 6 2 1 × 10¹ K. pneumoniae 7 AFR NDM-1 + OXA-1 + CTX-M-15 + >32 >32 >32 1 × 10¹ CMY-6 + TEM-1 K. pneumoniae IND NDM-1 + OXA-1 + CTX-M-15 1 8 4 1 × 10¹ E. coli GEN NDM-1 + OXA-1 + CMY-30 + 8 >32 12 1 × 10¹ TEM-1 E. coli RIC NDM-1 + OXA-1 + OXA-10 + 1 3 1 1 × 10¹ CMY-16 E. coli GUE NDM-1 + OXA-1 + TEM-1 3 3 2 1 × 10¹ E. coli AUS NDM-1 + CTX-M-15 + TEM-1 6 32 16 1 × 10¹ E. coli ALL NDM-1 + OXA-1 + OXA-2 + 4 >32 8 1 × 10¹ CTX-M-15 + TEM-1 E. coli IR5 NDM-1 + CTX-M-15 + TEM-1 16 >32 16 1 × 10¹ E. cloacae IR38 NDM-1 + CTX-M-15 2 16 2 1 × 10¹ P. stuartii NDM NDM-1 + OXA-1 + CMY-6 12 0.38 1.5 >5 × 10⁷  C. freundii STE NDM-1 + OXA-1 + OXA-9 + >32 >32 >32 1 × 10¹ OXA-10 + CTX-M-15 + TEM-1 KPC-2 E. coli PSP KPC-2 0.5 0.5 0.5 1 × 10² E. cloacae KPC-2 4 6 2 1 × 10¹ E. coli COL KPC-2 4 4 2 1 × 10¹ K. pneumoniae COL KPC-2 + TEM-1 + SHV-1 + CTX- 4 4 32 1 × 10¹ M-15 VIM or IMP E. coli MAD VIM-1 + CTX-M-3 1.5 0.38 0.5 1 × 10⁵ E. coli DIH VIM-19 8 16 4 1 × 10¹ K. pneumoniae MAD VIM-1 + CTX-M-3 1 0.5 1 1 × 10¹ E. coli JAP IMP-1 0.5 3 0.5 1 × 10⁶ K. pneumoniae TUR IMP-1 1 2 8 1 × 10⁶ Controls K. pneumoniae KPN^(b) CTX-M-15 0.12 0.12 0.12 >5 × 10⁷  E. cloacae CLO^(b) CTX-M-15 0.12 0.12 0.12 >6 × 10⁷  E. coli FOR^(b) CTX-M-15 0.12 0.12 0.12 >6 × 10⁷  E. coli E14^(b) CTX-M-14 0.12 0.12 0.12 >4 × 10⁷  E. cloacae CVB^(b) VEB-1 0.12 0.12 0.12 >5 × 10⁷  E. coli EVB^(b) VEB-1 0.12 0.12 0.12 >3 × 10⁷  P. mirabilis PMA^(c) ACC-1 0.25 0.12 0.12 >5 × 10⁷  E. coli ECA^(c) ACC-1 0.12 0.12 0.12 >5 × 10⁷  K. pneumoniae KDH^(c) DHA-2 0.12 0.5 0.12 >5 × 10⁷  E. coli MET^(c) Chromosome-encoded extended- 0.12 0.12 0.12 >5 × 10⁷  spectrum cephalosporinase E. coli SYD^(c) CMY-2 0.12 0.12 0.12 >5 × 10⁷  E. cloacae ARF^(d) AmpC 0.12 1 0.12 >5 × 10⁷  E. cloacae BLA^(d) AmpC 0.12 1 0.12 >5 × 10⁷  E. cloacae CON^(d) AmpC 0.12 1 0.12 >5 × 10⁷  E. cloacae AZA^(d) AmpC 0.12 1 0.12 >5 × 10⁷  K. pneumoniae MEK^(e) CTX-M-15 + SHV-11 1.5 >32 6 1 × 10¹ K. pneumoniae SIM^(e) CTX-M-15 + TEM-1 + SHV-1 8 >32 6 1 × 10¹ K. pneumoniae SHM^(e) CTX-M-15 + TEM-1 + SHV-11 3 >32 1 1 × 10¹ K. pneumoniae COO^(e) CTX-M-15 + SHV28 8 >32 4 1 × 10¹ K. pneumoniae FOS^(e) CTX-M-15 + TEM-1 + SHV-11 6 >32 >32 1 × 10² K. pneumoniae SHV-2a 0.25 2 0.38 1 × 10² 648236^(e) K. pneumoniae BER^(e) TEM-1 + SHV-28 1 4 1 1 × 10³ K. pneumoniae BED^(e) CTX-M-15 + TEM-1 + SHV-1 1.5 >32 4 1 × 10⁴ K. pneumoniae SHI^(e) CTX-M-15 + TEM-1 + SHV-1 0.25 1 1 7 × 10⁴ K. pneumoniae LEG^(e) CTX-M-15 + TEM-1 + SHV-12 0.75 >32 3 2 × 10⁴ K. pneumoniae ALE^(e) CTX-M-15 + SHV-1 1 >32 4 1 × 10⁵ C. freundii MAU^(e) Overexpressed AmpC + TEM-3 1 8 1 1 × 10⁵ ^(a)Abbreviations: IMP, imipenem; ETP, ertapenem; MP, meropenem. ^(b)Ertapenem susceptible isolates producing an ESBL. ^(c)Ertapenem susceptible isolates producing a high-level AmpC. ^(d)Reduced susceptibility to ertapenem due to overexpressed AmpC. ^(e)Reduced susceptibility to ertapenem due to porin deficiency. Beta-lactame of the ESBL type: CTX-M, TEM, SHV, VEB. Beta lactame of the High-level AmpC type: ACC, DHA, CMY.

The results above show that the Applicant has developed a new, reliable and highly sensitive culture medium for detecting carbapenemase-producing bacteria isolates from a collection of well-characterized strains, and preferably isolates with a reduced susceptibility to carbapenems. Almost all OXA-48, NDM-1 and KPC-type carbapenemase producers were detected with a low detection limit of 1×10¹ to 1×10² CFU/ml by using this medium. In fact, the specificity and sensitivity of the culture medium according to the invention is much higher than that of the ChromID ESBL and the CHROMagar-KPC media.

Example 2 Storage Stability of the SUPERCARBA Agar Culture Medium

To assess the storage stability of SUPERCARBA agar, E. cloacae ARF with over-expressed AmpC was sub-cultured daily onto plates from a single batch of SUPERCARBA Drigalski agar stored at 4° C. Growth of E. cloacae ARF was consistently inhibited on the SUPERCARBA agar throughout a seven-day period.

Example 3 Detection of Carbapenemase Producers in Enterobacteriaceae using a Novel Screening Medium

Abstract

A Drigalski agar-based culture medium containing ertapenem, cloxacillin, and zinc sulfate (SUPERCARBA medium) was tested for screening carbapenemase-producing Enterobacteriaceae. OXA-48 (n=44), NDM (n=25), VIM/IMP (n=27), and KPC producers (n=18) were detected with a low detection limit. Its overall sensitivity (95.6%) was higher than those of the currently available ChromID ESBL (bioMerieux) and the CHROMagar-KPC(CHROMagar) screening media. The SUPERCARBA medium provides a significative improvement for detection of the most common types of carbapenemase producers.

Taking into account the current importance of detecting carbapenemase producers with accuracy, we have designed a novel screening (termed SUPERCARBA) medium. The rationale for the design of this medium was that it should be able to detect carbapenemase producers with low-level resistance to carbapenems, and be as selective as possible by inhibiting growth of carbapenem-resistant but non-carbapenemase producing isolates.

Different concentrations of several carbapenem molecules were tested, and finally ertapenem was added to a Drigalski agar medium at a concentration of 0.25 μg/ml. ZnSO₄ (70 μg/ml) was added to improve expression of metallo-β-lactamases by MBL producers. Cloxacillin (250 μg/ml) which is a cephalosporinase (AmpC-type β-lactamase) inhibitor was used to prevent growth of isolates expressing high level expression of cephalosporinases, such as Enterobacter cloacae, Enterobacter aerogenes, Morganella morgannii, and Serratia marcescens. Those isolates are clinically-significant sources of carbapenem resistance when outer-membrane permeability defect is associated (Girlich, et al., 2009. Antimicrob. Agents Chemother. 53:832-834, Mammeri et al. 2008. FEMS Microbiol. Lett. 282:238-240).

One-hundred and fourteen carbapenemase-producing isolates belonging to various enterobacterial species of worldwide origin were included in the study, all having a β-lactamase content characterized at the molecular level (Table 2). The strains were as follows: KPC producers (n=18), VIM producers (n=12), IMP producers (n=15), NDM-1 producers (n=25), together with OXA-48-(n=41) and OXA-181 producers (n=3). Seventy-five of those isolates co-expressed an ESBL (Table 2). Strains that did not express any carbapenemase were used as controls, consisting in isolates showing reduced susceptibility to ertapenem due to an overexpressed AmpC (n=10), or to an ESBL (n=12), and/or porin deficiency. Wild-type ertapenem-susceptible isolates, restricted-spectrum β-lactamase producers, ESBL producers, and high-level AmpC producers were also included as controls (n=40) (Table 2). Using an inoculum of ˜2×10⁷ CFU/ml (range, 1.5×10⁷ to 3.5×10⁸ CFU/ml), serial 10-fold dilutions of the isolates were made in normal saline and 100 μl were plated onto the SUPERCARBA medium and compared to results obtained using CHROMagar KPC and ChromID ESBL. Viable bacteria were counted after 24 h of culture at 37° C. The sensitivity and specificity cut-off values were set at 1×10³ CFU/ml, i-e. a limit value of 1×10³ CFU/ml and above was considered as “not efficiently detected”.

The lowest limit of detection of OXA-48, OXA-181, NDM-1 and KPC producers ranged from 1×10¹ to 1×10² CFU/ml (Table 2). A single NDM producer (NDM-1-producing Providencia stuartii isolate (Poirel et al., 2011. Antimicrob. Agents Chemother. 55:5403-5407.) was not efficiently detected on the SUPERCARBA medium (limit of detection 1×10⁷ CFU/ml) (Table 2). Its lack of detection might be explained by its low MIC values of ertapenem (0.38 μg/ml) and a likely weak expression of the bla_(NDM-1) gene, related to chromosomal insertion of the bla_(NDM-1) gene. As expected, OXA-181-producing K. pneumoniae were also well detected with the SUPERCARBA medium. The lowest limit of detection of VIM and IMP producers ranged from 1×10¹ to 1×10⁶ CFU/ml (Table 1). Although addition of zinc sulfate decreased significantly to lower the detection limit for VIM and IMP producers, a few VIM and IMP producers were not efficiently detected on this medium (detection limit >1×10³ CFU/ml). As expected, growth of isolates that do not express any carbapenemase (i.e. AmpC and/or ESBL producers) was inhibited by the SUPERCARBA medium (with a detection limit much higher than 1×10³ CFU/ml). In particular, cloxacillin addition prevented growth of the isolates expressing cephalosporinases (Table 2). As previously shown, porin defect resulting in a decreased outer-membrane permeability leads to a reduced susceptibility to ertapenem of E. coli and K. pneumoniae. In this study, among the 19 ertapenem non-susceptible isolates, with MIC values of ertapenem being >0.25 μg/ml (1 Citrobacter freundii, 2 E. coli, 4 E. cloacae, and 12 K. pneumoniae isolates) and for which porin defect was involved in ertapenem resistance, 58% (n=11) were detected by selection on the SUPERCARBA medium (lower detection limit <10² CFU/ml) (Table 2). Addition of zinc sulfate and cloxacillin was useful for prevention of growth of many (up to 42%, n=8) of non-carbapenemase producing carbapenem-resistant isolates. Noticeably, non-carbapenemase-producing Acinetobacter baumannii and Pseudomonas aeruginosa, grew on the SUPERCARBA medium (data not shown). Similar results of growth of non-enterobacterial Gram negative rods were obtained using the ChromID ESBL and the CHROMagar KPC media (data not shown). These three media are suitable only for selection of Enterobacteriaceae.

A comparison of the results obtained with the ChromID ESBL and CHROMagar KPC media with those obtained with the SUPERCARBA medium showed that this latter screening medium is more efficient to detect carbapenemase-producing isolates (Tables 2 and 3). Indeed, sensitivity of the SUPERCARBA medium was 95.6%, higher than that of the ChromID ESBL (87.7%), and of the CHROMagar KPC (40.3%) medium. Moreover, the sensitivities of SUPERCARBA, determined for each class of carbapenemase producers, was higher (100%, 90% and 100% for classes A, B, and D, respectively) than those obtained for the two other screening media (Table 3). The specificity of the SUPERCARBA medium was also high (82.2%). A further improvement of the SUPERCARBA medium would be the addition of chromogenic molecules that would permit a species recognition.

To assess the storage ability of the SUPERCARBA medium, E. cloacae ARF that over-expressed AmpC was subcultured daily onto Drigalski plates from a single batch of SUPERCARBA medium stored at 4° C. Growth of this isolate was consistently inhibited on the SUPERCARBA agar during a seven-day period.

We propose here the very first screening medium that may detect not only KPC-, MBL-, but also OXA-48-producers. This medium represents a significative improvement as compared to the available screening media to detect carbapenemase producers, and particularly for detection of OXA-48 producers that do not co-express any ESBL. Taking into account that SUPERCARBA medium contains ertapenem at a low concentration, it may detect carbapenemase producers with low level resistance to carbapenems, which is a situation frequently observed for OXA-48 producers. In addition, this medium is useful for selecting specifically carbapenemase producers in stools that contain also a large amount of ESBL producers which growth will be inhibited. This property is particularly relevant, since high rates of ESBL carriage is now reported worldwide.

Modified SUPERCARBA media were prepared with similar results (see tables 4 to 11).

TABLE 2 Limit of detection of SUPERCARBA medium for 176 carbapenemase- and/or ESBL/AmpC-producing enterobacterial isolates as compared to those obtained with ChromID ESBL and CHROMagar KPC media. MIC values of imipenem, ertapenem and meropenem are provided for each strain. Lowest detection limit (CFU/ml) MIC (μg/ml) ChromID CHROMagar Strains -Lactamase content IPM^(a) ETP MEM SUPERCARBA ESBL KPC Ambler class A carbapenemases (KPC) K. pneumoniae 2303 KPC-2 ^(b) + SHV-11 >32 >32 >32 1 × 10¹ 1 × 10¹ 1 × 10¹ K. pneumoniae LIE KPC-2 + TEM-2 + OXA-9 >32 >32 >32 5 × 10¹ 1 × 10¹ 1 × 10¹ K. pneumoniae GES KPC-2 + TEM-1 + SHV-11 6 12 1.5 1 × 10¹ 1 × 10¹  1 × 10^(3 c) K. pneumoniae 588 KPC-2 + TEM-1 + SHV-11 + OXA-9 24 32 16 1 × 10¹ 1 × 10¹ 1 × 10¹ K. pneumoniae YC KPC-2 + TEM-1 + SHV-11 + SHV-12 + 4 24 2 1 × 10¹ 1 × 10¹ 1 × 10¹ OXA-9 K. pneumoniae A28006 KPC-2 + TEM-1 + CTX-M-2 + SHV-11 16 24 32 2 × 10¹ 1 × 10¹ 1 × 10¹ K. pneumoniae A33504 KPC-2 + TEM-1 + SHV-11 + CTX-M-2 + >32 >32 >32 1 × 10¹ 1 × 10¹ 1 × 10¹ OXA-9 K. pneumoniae MUS KPC-2 + TEM-1 + SHV-11 + SHV-12 0.75 4 1.5 1 × 10¹ 1 × 10¹ 1 × 10³ K. pneumoniae KAM KPC-3 + TEM-1 + SHV-11 8 12 2 1 × 10¹ 1 × 10¹ 5 × 10³ E. coli PSP KPC-2 + TEM-1 + OXA-1 0.5 0.5 0.5 1 × 10² 1 × 10¹ 1 × 10⁴ E. coli DIN KPC-2 + TEM-1 + OXA-1 1 >32 0.5 1 × 10¹ 1 × 10¹ 1 × 10¹ E. coli COL KPC-2 + TEM-1 + CTX-M-9 4 4 2 1 × 10¹ 1 × 10¹ 1 × 10³ E. coli LIL KPC-2 + TEM-1 + OXA-9 2 1.5 1 1 × 10¹ 1 × 10¹ 1 × 10¹ E. cloacae HMG KPC-2 + TEM-1 24 >32 16 1 × 10² 1 × 10¹ 1 × 10¹ E. cloacae CFVL KPC-2 + TEM-3 4 2 1 1 × 10¹ 1 × 10¹ 5 × 10⁵ E. cloacae HPTU KPC-2 + TEM-1 + SHV-11 2 4 1.5 1 × 10¹ 1 × 10¹ 1 × 10¹ S. marcescens D6403 KPC-2 + TEM-1 >32 >32 >32 1 × 10¹ 1 × 10¹ 1 × 10¹ S. marcescens C7052 KPC-2 + TEM-1 + SHV-12 >32 >32 >32 1 × 10¹ 1 × 10¹ 1 × 10¹ Ambler class B carbapenemases K. pneumoniae OMA419 NDM-1 + OXA-1 1.5 6 2 1 × 10¹ 1 × 10¹ 1 × 10² K. pneumoniae KI2 NDM-1 + CTX-M-15 + OXA-1 1 8 4 1 × 10¹ 1 × 10¹ 1 × 10¹ K. pneumoniae UK NDM-1 + CTX-M-15 + CMY-4 + OXA-1 >32 >32 >32 1 × 10¹ 1 × 10¹ 1 × 10¹ K. pneumoniae 6642 GEN NDM-1 + CTX-M-15 + OXA-1 + OXA-10 1 16 3 1 × 10¹ 1 × 10¹ 1 × 10¹ K. pneumoniae 6759 GEN NDM-1 + CTX-M-15 + CMY-16 + OXA-1 + 12 >32 >32 1 × 10¹ 1 × 10¹ 1 × 10¹ OXA-9 + OXA-10 K. pneumoniae OMA601 NDM-1 + CTX-M-15 + OXA-1 + OXA-9 >32 >32 >32 1 × 10¹ 1 × 10¹ 1 × 10¹ K. pneumoniae 7AFR NDM-1 + TEM-1 + CTX-M-15 + CMY-6 + >32 >32 >32 1 × 10¹ 1 × 10¹ 1 × 10¹ OXA-1 K. pneumoniae OM2 NDM-1 + TEM-1 + CTX-M-3 + SHV-11 + 0.75 8 1.5 1 × 10¹ 1 × 10¹ 3 × 10⁴ OXA-1 K. pneumoniae OM4 NDM-1 + TEM-1 + CTX-M-15 + SHV-12 + 4 >32 16 1 × 10¹ 1 × 10¹ 1 × 10¹ OXA-9 K. pneumoniae OM8 NDM-1 + TEM-1 + CTX-M-15 + SHV-11 + 2 >32 4 2 × 10¹ 1 × 10¹ 1 × 10² OXA-1 K. pneumoniae OM13 NDM-1 + TEM-1 + CTX-M-15 + SHV-28 + 3 4 2 1 × 10¹ 1 × 10¹ 3 × 10⁴ OXA-1 + OXA-9 K. pneumoniae OM15 NDM-1 + CTX-M-15 + SHV-130 + OXA-1 1.5 12 3 1 × 10¹ 1 × 10¹ 3 × 10⁵ K. pneumoniae OM16 NDM-1 + CTX-M-15 + OXA-1 + OXA-181 8 >32 16 3 × 10¹ 1 × 10¹ 1 × 10¹ K. pneumoniae OM19 NDM-1 + CTX-M-15 + SHV-12 + OXA-1 4 24 8 1 × 10¹ 1 × 10¹ 4 × 10² K. pneumoniae KIE NDM-1 + SHV-38 + CMY-16 + OXA-10 0.75 2 1 1 × 10¹ 1 × 10¹ 1 × 10⁴ E. coli GUE NDM-1 + TEM-1 + OXA-1 3 3 2 1 × 10¹ 1 × 10¹ 1 × 10⁵ E. coli AUS NDM-1 + TEM-1 + CTX-M-15 6 32 16 1 × 10¹ 1 × 10¹ 1 × 10¹ E. coli IR5 NDM-1 + TEM-1 + CTX-M-15 16 >32 16 1 × 10¹ 1 × 10¹ 1 × 10¹ E. coli GEN NDM-1 + TEM-1 + CMY-30 + OXA-1 8 >32 12 1 × 10¹ 1 × 10¹ 1 × 10¹ E. coli RIC NDM-1 + CMY-16 + OXA-1 + OXA-10 1 3 1 1 × 10¹ 1 × 10¹ 1 × 10⁵ E. coli ALL NDM-1 + TEM-1 + CTX-M-15 + OXA-1 + 4 >32 8 1 × 10¹ 1 × 10¹ 1 × 10¹ OXA-2 E. coli OM20 NDM-1 + TEM-1 + CTX-M-15 2 >32 8 1 × 10¹ 1 × 10¹ 1 × 10¹ E. cloacae IR38 NDM-1 + CTX-M-15 2 16 2 1 × 10¹ 3 × 10² 4 × 10⁴ P. stuartii PS1 NDM-1 + CMY-6 + OXA-1 12 0.38 1.5 1 × 10⁷ 1 × 10³ 1 × 10⁷ C. freundii STE NDM-1 + TEM-1 + CTX-M-15 + VIM-4 + >32 >32 >32 1 × 10¹ 1 × 10¹ 1 × 10¹ OXA-1 + OXA-9 + OXA-10 + OXA-181 K. pneumoniae 0404024 VIM-1 >32 >32 >32 1 × 10¹ 1 × 10¹ 1 × 10¹ K. pneumoniae 0511135 VIM-1+ SHV-12 >32 >32 >32 1 × 10¹ 1 × 10¹ 1 × 10¹ K. pneumoniae 0404020 VIM-1+ SHV-5 >32 >32 >32 1 × 10¹ 1 × 10¹ 1 × 10¹ K. pneumoniae ENN VIM-1+ SHV-5 0.5 1.5 0.38 1 × 10¹ 1 × 10¹ 1 × 10¹ K. pneumoniae MAD VIM-1+ CTX-M-3 1 0.5 1 1 × 10¹ 1 × 10¹ 2 × 10⁴ E. coli DIH VIM-19 8 16 4 1 × 10¹ 1 × 10¹ 1 × 10¹ E. coli 0404018 VIM-1+ CMY-6 3 1.5 1 5 × 10¹ 1 × 10¹ >1 × 10⁸   E. coli 1008077 VIM-1+ TEM-1 + CTX-M-15 >32 4 4 1 × 10¹ 1 × 10¹ >1 × 10⁸   E. coli MAD VIM-1+ CTX-M-3 1.5 0.38 0.5 1 × 10⁵ 1 × 10¹ 2 × 10⁵ E. cloacae KAR VIM-1+ SHV-70 1 0.38 0.5 1 × 10⁶ 1 × 10¹ >1 × 10⁸   E. cloacae 1008029 VIM-1+ CTX-M-3 >32 >32 >32 2 × 10¹ 1 × 10¹ 1 × 10¹ S. marcescens 1008091 VIM-1+ CTX-M-15 >32 >32 >32 1 × 10¹ 1 × 10¹ 1 × 10¹ K. pneumoniae TUR IMP-1 1 2 8 1 × 10⁶ 2 × 10¹ 1 × 10¹ K. pneumoniae 0709121 IMP-1 1.5 3 1 1 × 10¹ 1 × 10¹ 1 × 10³ K. pneumoniae 0709124 IMP-1 + TEM-15 8 3 2 1 × 10¹ 1 × 10¹ 1 × 10⁴ K. pneumoniae 0709125 IMP-1 + TEM-1 + SHV-12 1.5 4 2 1 × 10¹ 1 × 10¹ 1 × 10³ K. pneumoniae 0709127 IMP-1 + TEM-1 0.5 4 1 1 × 10¹ 1 × 10¹ 1 × 10⁴ K. pneumoniae TWA IMP-8 1 1 0.5 1 × 10¹ 1 × 10¹ >1 × 10⁸   K. pneumoniae TAW IMP-8 + SHV-12 0.5 0.5 0.5 4 × 10² 1 × 10¹ >1 × 10⁸   E. coli JAP IMP-1 0.5 3 0.5 1 × 10⁴ 1 × 10¹ 2 × 10⁵ E. coli TWA IMP-8 + SHV-12 6 8 3 1 × 10¹ 1 × 10¹ 1 × 10¹ E. coli 1108013 IMP-1 + TEM-1 0.5 4 1 1 × 10¹ 1 × 10¹ 1 × 10⁶ E. cloacae TWA IMP-8 1.5 1 1 1 × 10¹ 1 × 10¹ 1 × 10² E. cloacae TAW IMP-8 + SHV-12 0.75 0.5 0.5 1 × 10² 1 × 10¹ >1 × 10⁸   E. cloacae 1008079 IMP-1 8 >32 >32 1 × 10¹ 1 × 10² 1 × 10⁷ E. cloacae 1008187 IMP-1 + CTX-M-15 8 >32 4 1 × 10¹ 1 × 10¹ 1 × 10⁴ S. marcescens 0911033 IMP-1 >32 >32 >32 1 × 10¹ 1 × 10¹ 1 × 10¹ Ambler Class D Carbapenemases K. pneumoniae BIC OXA-48 0.5 2 0.5 1 × 10¹ >1 × 10⁸   5 × 10⁶ K. pneumoniae BEL OXA-48 1 4 1 1 × 10¹ >1 × 10⁸   1 × 10⁶ K. pneumoniae RAM OXA-48 1 4 1 1 × 10¹ >1 × 10⁸   1 × 10⁵ K. pneumoniae LIB OXA-48 16 16 16 1 × 10¹ >1 × 10⁸   5 × 10⁴ K. pneumoniae BOU OXA-48 0.38 0.5 0.25 1 × 10¹ >1 × 10⁸   1 × 10⁸ K. pneumoniae SCO OXA-48 0.5 0.75 0.25 1 × 10¹ >1 × 10⁸   >1 × 10⁸   K. pneumoniae LOU OXA-48 4 16 0.5 1 × 10¹ >1 × 10⁸   >1 × 10⁸   K. pneumoniae TIK OXA-48 0.75 2 0.38 1 × 10¹ >1 × 10⁸   >1 × 10⁸   K. pneumoniae OM14 OXA-48 + TEM-1 0.5 1 0.38 1 × 10¹ >1 × 10⁸   5 × 10⁷ K. pneumoniae CHA OXA-48 + TEM-1 0.38 1 0.5 1 × 10¹ >1 × 10⁸   >1 × 10⁸   K. pneumoniae EGY OXA-48 + CTX-M-15 2 3 2 1 × 10¹ 2 × 10¹ 1 × 10⁵ K. pneumoniae ROU OXA-48 + CTX-M-15 0.5 1.5 0.25 1 × 10¹ 1 × 10¹ >1 × 10⁸   K. pneumoniae BEY OXA-48 + TEM-1 + CTX-M-15 0.38 0.38 0.38 5 × 10² 1 × 10¹ 1 × 10⁸ K. pneumoniae DAL OXA-48 + TEM-1 + CTX-M-15 0.38 2 0.38 1 × 10¹ 1 × 10¹ 4 × 10⁵ K. pneumoniae BAJ OXA-48 + TEM-1 + CTX-M-15 + SHV-28 0.5 1.5 0.38 1 × 10¹ 1 × 10¹ >1 × 10⁸   K. pneumoniae BEN OXA-48 + TEM-1 + CTX-M-15 + SHV-28 0.38 1 0.25 1 × 10¹ 1 × 10¹ >1 × 10⁸   K. pneumoniae DUW OXA-48 + TEM-1 + CTX-M-15 + SHV-28 32 32 32 1 × 10¹ 1 × 10¹ 1 × 10¹ K. pneumoniae SIC OXA-48 + CTX-M-15 + SHV-28 0.25 1 0.25 1 × 10¹ 1 × 10¹ >1 × 10⁸   K. pneumoniae AEL OXA-48 + CTX-M-15 + SHV-28 + OXA-1 0.5 6 0.38 1 × 10¹ 1 × 10¹ 5 × 10² K. pneumoniae AMS OXA-48 + TEM-1 + CTX-M-15 + OXA-1 0.5 2 0.38 1 × 10¹ 1 × 10¹ >1 × 10⁸   K. pneumoniae ELK OXA-48 + TEM-1 + CTX-M-15 + SHV-11 0.5 3 0.38 1 × 10¹ 1 × 10¹ >1 × 10⁸   K. pneumoniae VER OXA-48 + TEM-1 + CTX-M-15 + SHV-11 0.38 2 0.38 1 × 10¹ 1 × 10¹ >1 × 10⁸   K. pneumoniae VSG OXA-48 + TEM-1 + CTX-M-15 + OXA-1 0.75 3 0.75 1 × 10¹ 1 × 10¹ >1 × 10⁸   K. pneumoniae HPA OXA-48 + TEM-1 + CTX-M-15 + OXA-1 1.5 >32 12 1 × 10¹ 1 × 10¹ >1 × 10⁸   K. pneumoniae OM11 OXA-48 + TEM-1 + CTX-M-14 0.5 0.75 0.25 1 × 10¹ 1 × 10¹ 5 × 10⁷ K. pneumoniae DIA OXA-48 + TEM-1b + CTX-M-15 + SHV-11 + >32 >32 >32 1 × 10¹ 1 × 10¹ 1 × 10¹ OXA-1 E. coli ROB OXA-48 0.5 0.75 0.25 2 × 10¹ >1 × 10⁸   >1 × 10⁸   E. coli HAN OXA-48 + CTX-M-15 3 16 1 5 × 10¹ 1 × 10¹ 3 × 10⁴ E. coli BOU OXA-48 + CTX-M-15 0.5 0.75 0.125 2 × 10¹ 1 × 10¹ >1 × 10⁸   E. coli OM3 OXA-48 + TEM-1 + CTX-M-15 0.5 1 0.38 1 × 10¹ 1 × 10¹ >1 × 10⁸   E. coli OM22 OXA-48 + TEM-1 + CTX-M-15 0.5 1 0.25 1 × 10¹ 1 × 10¹ >1 × 10⁸   E. coli BER OXA-48 + TEM-1 + CTX-M-15 0.38 1.5 0.19 5 × 10¹ 1 × 10¹ >1 × 10⁸   E. coli AME OXA-48 + CTX-M-24 0.25 0.5 0.19 2 × 10¹ 1 × 10¹ >1 × 10⁸   E. coli ZAN OXA-48 + TEM-1 + CTX-M-14 0.38 8 0.75 1 × 10¹ 1 × 10¹ >1 × 10⁸   E. coli BON OXA-48 + TEM-1 + CTX-M-24 0.38 0.5 0.19 1 × 10¹ 1 × 10¹ >1 × 10⁸   E. coli BOK OXA-48 + CTX-M-15 0.25 0.38 0.19 2 × 10¹ 1 × 10¹ >1 × 10⁸   E. cloacae TUR OXA-48 + SHV-5 0.5 0.5 0.5 1 × 10¹ 2 × 10¹ 1 × 10⁷ E. cloacae 501 OXA-48 + TEM-1 + CTX-M-15 1 16 1.5 1 × 10¹ 1 × 10¹ 1 × 10¹ E. cloacae BEU OXA-48 + TEM-1 + CTX-M-15 + SHV-12 0.5 8 0.5 1 × 10¹ 1 × 10¹ 1 × 10⁴ C. koseri ROU OXA-48 0.38 2 0.38 1 × 10¹ >1 × 10⁸   >1 × 10⁸   C. koseri VER OXA-48 0.75 2 0.38 1 × 10¹ >1 × 10⁸   >1 × 10⁸   K. pneumoniae HOL OXA-181 + CTX-M-15 1 4 1 1 × 10¹ 1 × 10¹ 1 × 10¹ K. pneumoniae OMA OXA-181 + CTXM-15 + OXA-1 0.5 2 0.5 1 × 10¹ 1 × 10¹ >1 × 10⁸   P. rettgeri RAP OXA-181 + OXA-1 8 1 2 5 × 10² 1 × 10¹ 1 × 10¹ Non-carbapenemase producers K. pneumoniae 7725 SHV-1 0.19 0.006 0.032 >1 × 10⁸   >1 × 10⁸   >1 × 10⁸   K. pneumoniae 0227 SHV-1 0.19 0.008 0.016 >1 × 10⁸   >1 × 10⁸   >1 × 10⁸   K. pneumoniae 648236^(d) SHV-2a 0.25 2 0.38 1 × 10² 1 × 10¹ >1 × 10⁸   K. pneumoniae 1022 SHV-2a + SHV-28 0.5 0.016 0.023 >1 × 10⁸   1 × 10¹ >1 × 10⁸   K. pneumoniae BER^(d) SHV-28 + TEM-1 1 4 1 1 × 10² 1 × 10³ 1 × 10³ K. pneumoniae KPN CTX-M-15 0.12 0.012 0.012 1 × 10⁷ 1 × 10¹ >1 × 10⁸   K. pneumoniae 10112 CTX-M-15 + TEM-1 + SHV-11 0.5 0.016 0.023 6 × 10⁷ 1 × 10¹ >1 × 10⁸   K. pneumoniae 1025 CTX-M-14 + TEM-1 + SHV-11 0.12 0.016 0.016 >1 × 10⁸   1 × 10¹ >1 × 10⁸   K. pneumoniae MEK^(d) CTX-M-15 + SHV-11 1.5 >32 6 1 × 10¹ 1 × 10¹ 1 × 10¹ K. pneumoniae SIM^(d) CTX-M-15 + TEM-1 + SHV-1 8 >32 6 1 × 10¹ 1 × 10¹ 1 × 10² K. pneumoniae SHM^(d) CTX-M-15 + TEM-1 + SHV-11 3 >32 3 1 × 10¹ 1 × 10¹ 1 × 10¹ K. pneumoniae COO^(d) CTX-M-15 + SHV-28 8 >32 4 1 × 10¹ 1 × 10¹ 1 × 10¹ K. pneumoniae FOS^(d) CTX-M-15 + TEM-1 + SHV-11 6 >32 >32 1 × 10² 1 × 10¹ 1 × 10¹ K. pneumoniae BED^(d) CTX-M-15 + TEM-1 + SHV-11 1.5 >32 4 1 × 10¹ 1 × 10¹ 1 × 10¹ K. pneumoniae SHI^(d) CTX-M-15 + TEM-1 + SHV-11 0.25 1 1 7 × 10⁴ 1 × 10¹ >1 × 10⁸   K. pneumoniae LEG^(d) CTX-M-15 + TEM-1 + SHV-12 0.75 >32 3 2 × 10⁴ 2 × 10¹ 2 × 10¹ K. pneumoniae ALE^(d) CTX-M-15 + SHV-1 1 >32 4 1 × 10⁵ 1 × 10¹ 1 × 10¹ K. pneumoniae KDH^(e) DHA-2 0.12 0.5 0.12 1 × 10² 1 × 10¹ >1 × 10⁸   E. coli 6252 (wild type) 0.12 0.004 0.008 >1 × 10⁸   >1 × 10⁸   >1 × 10⁸   E. coli 6367 (wild type) 0.19 0.006 0.012 >1 × 10⁸   >1 × 10⁸   >1 × 10⁸   E. coli 1082 TEM-1 0.19 0.019 0.016 >1 × 10⁸   >1 × 10⁸   >1 × 10⁸   E. coli 1034 TEM-1 + SHV-38 0.19 0.006 0.016 >1 × 10⁸   >1 × 10⁸   >1 × 10⁸   E. coli 1048 TEM-1 + SHV-2a 0.19 0.012 0.016 >1 × 10⁸   1 × 10¹ >1 × 10⁸   E. coli 1008 CTX-M-1 + TEM-1 0.19 0.016 0.016 >1 × 10⁸   1 × 10¹ >1 × 10⁸   E. coli 10122 CTX-M-1 + TEM-1 0.19 0.016 0.016 >1 × 10⁸   1 × 10¹ >1 × 10⁸   E. coli 1020 CTX-M-1 + TEM-1 0.19 0.023 0.016 >1 × 10⁸   1 × 10¹ >1 × 10⁸   E. coli 10121 CTX-M-2 0.19 0.016 0.016 >1 × 10⁸   1 × 10¹ >1 × 10⁸   E. coli 1023 CTX-M-2 + TEM-1 0.12 0.016 0.016 >1 × 10⁸   1 × 10¹ >1 × 10⁸   E. coli E14 CTX-M-14 0.12 0.012 0.012 >1 × 10⁸   1 × 10¹ >1 × 10⁸   E. coli FOR CTX-M-15 0.12 0.012 0.012 >1 × 10⁸   1 × 10¹ >1 × 10⁸   E. coli 1033 CTX-M-15 0.19 0.012 0.016 >1 × 10⁸   1 × 10¹ >1 × 10⁸   E. coli EVB VEB-1 0.12 0.012 0.012 >1 × 10⁸   1 × 10¹ >1 × 10⁸   E. coli 1092 OXA-1 0.12 0.19 0.023 >1 × 10⁸   1 × 10¹ >1 × 10⁸   E. coli ECA ACC-1 0.12 0.012 0.012 >1 × 10⁸   5 × 10³ >1 × 10⁸   E. coli SYD CMY-2 0.12 0.012 0.012 >1 × 10⁸   1 × 10¹ >1 × 10⁸   E. coli MET Chromosome-encoded extended- 0.12 0.012 0.012 >1 × 10⁸   1 × 10¹ >1 × 10⁸   spectrum cephalosporinase E. coli MAR^(e) Overexpressed AmpC 16 >32 2 1 × 10² 1 × 10¹ 1 × 10¹ E. coli HB4^(d) (OmpC-, OmpF-) 0.12 1 0.25 1 × 10¹ >1 × 10⁸   >1 × 10⁸   E. aerogenes 1009 TEM-24 0.19 0.12 0.016 >1 × 10⁸   1 × 10¹ >1 × 10⁸   E. aerogenes 1085 TEM-24 0.12 0.19 0.023 >1 × 10⁸   1 × 10¹ >1 x 10⁸   E. cloacae 7746 (wild type) 0.38 0.064 0.032 >1 × 10⁸   >1 × 10⁸   >1 × 10⁸   E. cloacae 7725 (wild type) 0.19 0.008 0.012 >1 × 10⁸   >1 × 10⁸   >1 × 10⁸   E. cloacae 5434 (wild type) 0.38 0.016 0.032 >1 × 10⁸   >1 × 10⁸   >1 × 10⁸   E. cloacae 1012 TEM-1 + SHV-12 0.19 0.016 0.016 >1 × 10⁸   1 × 10¹ >1 × 10⁸   E. cloacae 1072^(e) TEM-1 + OXA-1 0.38 0.5 0.064 >1 × 10⁸   1 × 10¹ >1 × 10⁸   E. cloacae CLO CTX-M-15 0.12 0.12 0.12 1 × 10⁷ 1 × 10¹ >1 × 10⁸   E. cloacae 10111^(e) TEM-1 + CTX-M-15 0.5 0.75 0.094 >1 × 10⁸   1 × 10¹ >1 × 10⁸   E. cloacae 1027 TEM-1 + CTX-M-15 0.19 0.016 0.016 >1 × 10⁸   1 × 10¹ >1 × 10⁸   E. cloacae CVB VEB-1 0.12 0.12 0.12 1 × 10⁴ 1 × 10¹ >1 × 10⁸   E. cloacae 1019^(e) TEM-1 0.25 1 0.094 >1 × 10⁸   1 × 10¹ >1 × 10⁸   E. cloacae ARF^(e) Overexpressed AmpC 0.12 1 0.12 1 × 10⁷ 1 × 10¹ >1 × 10⁸   E. cloacae BLA^(e) Overexpressed AmpC 0.12 1 0.12 1 × 10⁷ 1 × 10¹ >1 × 10⁸   E. cloacae CON^(e) Overexpressed AmpC 0.25 4 0.25 1 × 10⁷ 1 × 10¹ >1 × 10⁸   E. cloacae AZA^(e) Overexpressed AmpC 0.12 1 0.12 1 × 10⁷ 1 × 10⁶ >1 × 10⁸   C. freundii 7767 (wild type) 0.25 0.008 0.016 >1 × 10⁸   >1 × 10⁸   >1 × 10⁸   C. freundii 10107 TEM-1 + SHV-12 0.38 0.016 0.023 >1 × 10⁸   1 × 10¹ >1 × 10⁸   C. freundii 1003 CTX-M-15 + TEM-1 0.38 0.016 0.023 >1 × 10⁸   1 × 10¹ >1 × 10⁸   C. freundii 10135 CTX-M-15 0.38 0.016 0.023 >1 × 10⁸   1 × 10¹ >1 × 10⁸   C. freundii MAU^(e) Overexpressed AmpC + TEM-3 1 8 1 1 × 10⁵ 1 × 10¹ 1 × 10⁵ S. typhimurium 1081 CTX-M-1 0.25 0.19 0.032 >1 × 10⁸   1 × 10¹ >1 × 10⁸   P. mirabilis 1031 CTX-M-14 + TEM-1 + SHV-11 1.5 0.047 0.032 >1 × 10⁸   1 × 10¹ >1 × 10⁸   P. mirabilis PMA ACC-1 0.25 0.094 0.064 >1 × 10⁸   >1 × 10⁸   >1 × 10⁸   ^(a)Abbreviations: IMP, imipenem; ETP, ertapenem; MP, meropenem. ^(b)Boldened β-lactamase name correspond to carbapenemase. ^(c) Underligned CFU counts are considered as negative results (cut off values set at ≧1 × 10³ CFU/ml) ^(d)Reduced susceptibility to ertapenem due to porin deficiency. ^(e)Reduced susceptibility to ertapenem due to overexpressed AmpC

TABLE 3 Sensitivity and specificity of SUPERCARBA, ChromID ESBL, and CHROMagar KPC media^(a) SUPER ChromID CHROMagar CARBA ESBL KPC SN (%)^(a) 95.6 87.7 40.3 SP (%) 82.2 24.2 85.5 SN class A^(b) 100 100 66.7 SN class B 90 98 55.8 SN class D 100 70 13.6 ^(a)Abbreviations: SN, sensitivity; SP, specificity. ^(b)Sensitivity was determined for each Ambler class of carbapenemase: class A are of KPC-type, class B of VIM, IMP, and NDM-type, whereas class D are of OXA-48-type.

TABLE 4 Limit of detection of SUPERCARBA medium for 21 carbapenemase- and/or ESBL/AmpC-producing enterobacterial isolates as compared to those obtained with modified-SUPERCARBA, i.e. with Trypticase-soy agar supplemented with vancomycin (20 μg/ml) and amphotericin B (30 μg/ml) replacing Drigalski agar. MIC values of imipenem, ertapenem and meropenem are provided for each strain. Lowest detection limit (CFU/ml) ETP TSA-Vanco- MIC (μg/ml) Cloxa -Zinc Ampho-modified Strains -Lactamase content IPM^(a) ETP MEM SUPERCARBA SUPERCARBA Ambler class A carbapenemases K. pneumoniae MUS KPC-2 + TEM-1 + SHV-11 + 0.75 4 1.5 1 × 10¹ 1 × 10¹ SHV-12 E. cloacae HPTU KPC-2 + TEM-1 + SHV-11 2 4 1.5 1 × 10¹ 1 × 10¹ Ambler class B carbapenemases K. pneumoniae NDM-1 + OXA-1 1.5 6 2 1 × 10¹ 1 × 10¹ OMA419 K. pneumoniae OM2 NDM-1 + TEM-1 + CTX-M-3 + 0.75 8 1.5 1 × 10¹ 1 × 10¹ SHV-11 + OXA-1 P. stuartii PS1 NDM-1 + CMY-6 + OXA-1 12 0.38 1.5 1 × 10⁷ 1 × 10³ E. coli JAP IMP-1 0.5 3 0.5 1 × 10⁴ 2 × 10¹ E. cloacae TAW IMP-8 + SHV-12 0.75 0.5 0.5 1 × 10² 2 × 10² Ambler class D carbapenemases K. pneumoniae SCO OXA-48 0.5 0.75 0.25 1 × 10¹ 1 × 10¹ K. pneumoniae BEN OXA-48 + TEM-1 + CTX-M-15 + 0.38 1 0.25 1 × 10¹ 1 × 10¹ SHV-28 E. coli ROB OXA-48 0.5 0.75 0.25 2 × 10¹ 1 × 10¹ E. coli AME OXA-48 + CTX-M-24 0.25 0.5 0.19 2 × 10¹ 1 × 10¹ E. cloacae TUR OXA-48 + SHV-5 0.5 0.5 0.5 1 × 10¹ 1 × 10¹ C. koseri VER OXA-48 0.75 2 0.38 1 × 10¹ 1 × 10¹ K. pneumoniae HOL OXA-181 + CTX-M-15 1 4 1 1 × 10¹ 1 × 10² P. rettgeri RAP OXA-181 + OXA-1 8 1 2 5 × 10² 1 × 10¹ Non-carbapenemase producers K. pneumoniae 7725 SHV-1 0.19 0.006 0.032 >1 × 10⁸   >1 × 10⁸   K. pneumoniae 648236^(d) SHV-2a 0.25 2 0.38 1 × 10² 3 × 10¹ E. coli 1034 TEM-1 + SHV-38 0.19 0.006 0.016 >1 × 10⁸   >1 × 10⁸   E. coli 1008 TEM-1 + CTX-M-1 0.19 0.016 0.016 >1 × 10⁸   >1 × 10⁸   E. coli HB4^(d) (OmpC-, OmpF-) 0.12 1 0.25 1 × 10¹ 1 × 10¹ C. freundii MAU^(e) Overexpressed AmpC + TEM-3 1 8 1 1 × 10⁵ 1 × 10¹

TABLE 5 Sensitivity and specificity of SUPERCARBA and SUPERCARBA- modified with TSA-Vanco-Ampho replacing Drigalski agar Detection medium Drig- ETP TSA-Vanco- Ampho- Cloxa -Zinc modified SUPERCARBA SUPERCARBA SN (%)^(a) 86.7 93.3 SP (%) 66.7 50 ^(a)Abbreviations: SN, sensitivity; SP, specificity.

TABLE 6 Limit of detection of SUPERCARBA medium for 40 carbapenemase- and/or ESBL/AmpC-producing enterobacterial isolates as compared to those obtained with modified-SUPERCARBA, i.e. with doripenem 0.012, 0.024 or 0.048 μg/ml replacing ertapenem. MIC values of imipenem, ertapenem and meropenem are provided for each strain. Lowest detection limit (CFU/ml) ETP DORI DORI DORI Cloxa 0.048 0.024 0.012 MIC (μg/ml) Zinc Cloxa Cloxa Cloxa Strains β-Lactamase content IPM^(a) ETP MEM (SUPERCARBA) Zinc Zinc Zinc Ambler class B carbapenemases E. coli JAP IMP-1 0.5 3 0.5 1 × 10⁴ 8 × 10⁴ 8 × 10⁴ 4 × 10² E. cloacae TAW IMP-8 + SHV-12 0.75 0.5 0.5 1 × 10² 1 × 10² 3 × 10¹ 1 × 10¹ Ambler class D carbapenemases K. pneumoniae BEL OXA-48 1 4 1 1 × 10¹ 2 × 10¹ 1 × 10¹ 1 × 10¹ K. pneumoniae SCO OXA-48 0.5 0.75 0.25 1 × 10¹ 1 × 10¹ 1 × 10¹ 1 × 10¹ K. pneumoniae ROU OXA-48 + CTX-M-15 0.5 1.5 0.25 1 × 10¹ 1 × 10⁴ 1 × 10¹ 4 × 10⁴ K. pneumoniae BAJ OXA-48 + TEM-1 + CTX-M-15 + 0.5 1.5 0.38 1 × 10¹ 1 × 10¹ 1 × 10¹ 2 × 10¹ SHV-28 K. pneumoniae BEN OXA-48 + TEM-1 + CTX-M-15 + 0.38 1 0.25 1 × 10¹ 2 × 10¹ 1 × 10¹ 1 × 10¹ SHV-28 K. pneumoniae SIC OXA-48 + CTX-M-15 + SHV-28 0.25 1 0.25 1 × 10¹ 2 × 10² 1 × 10² 1 × 10¹ K. pneumoniae AMS OXA-48 + TEM-1 + CTX-M-15 + 0.5 2 0.38 1 × 10¹ 2 × 10¹ 1 × 10¹ 2 × 10¹ OXA-1 K. pneumoniae ELK OXA-48 + TEM-1 + CTX-M-15 + 0.5 3 0.38 1 × 10¹ 2 × 10² 1 × 10¹ 2 × 10¹ SHV-11 K. pneumoniae VSG OXA-48 + TEM-1 + CTX-M-15 + 0.75 3 0.75 1 × 10¹ 2 × 10¹ 2 × 10¹ 1 × 10¹ OXA-1 E. coli ROB OXA-48 0.5 0.75 0.25 2 × 10¹ 2 × 10¹ 1 × 10¹ 1 × 10¹ E. coli BOU OXA-48 + CTX-M-15 0.5 0.75 0.125 2 × 10¹ 1 × 10⁴ 1 × 10¹ 1 × 10¹ E. coli AME OXA-48 + CTX-M-24 0.25 0.5 0.19 2 × 10¹ 2 × 10⁶ 2 × 10¹ 1 × 10¹ E. coli BON OXA-48 + TEM-1 + CTX-M-24 0.38 0.5 0.19 1 × 10¹ 2 × 10⁵ 2 × 10¹ 1 × 10¹ E. cloacae TUR OXA-48 + SHV-5 0.5 0.5 0.5 1 × 10¹ 1 × 10¹ 1 × 10¹ 1 × 10¹ C. koseri VER OXA-48 0.75 2 0.38 1 × 10¹ 3 × 10² 3 × 10¹ 1 × 10¹ K. pneumoniae HOL OXA-181 + CTX-M-15 1 4 1 1 × 10¹ 1 × 10¹ 1 × 10¹ 1 × 10² K. pneumoniae OMA OXA-181 + CTXM-15 + OXA-1 0.5 2 0.5 1 × 10¹ 1 × 10¹ 1 × 10¹ 4 × 10¹ Non-carbapenemase producers K. pneumoniae 7725 SHV-1 0.19 0.006 0.032 >1 × 10⁸   >1 × 10⁸   >1 × 10⁸   2 × 10² K. pneumoniae 648236^(d) SHV-2a 0.25 2 0.38 1 × 10² 1 × 10¹ 1 × 10¹ 2 × 10¹ K. pneumoniae BER^(d) SHV-28 + TEM-1 1 4 1 1 × 10² 1 × 10² 1 × 10² 1 × 10¹ K. pneumoniae 1025 CTX-M-14 + TEM-1 + SHV-11 0.12 0.016 0.016 >1 × 10⁸   >1 × 10⁸   >1 × 10⁸   1 × 10¹ K. pneumoniae KDH^(e) DHA-2 0.12 0.5 0.12 1 × 10² 1 × 10¹ 1 × 10¹ 1 × 10¹ E. coli 1034 TEM-1 + SHV-38 0.19 0.006 0.016 >1 × 10⁸   >1 × 10⁸   >1 × 10⁸   1 × 10¹ E. coli 1048 TEM-1 + SHV-2a 0.19 0.012 0.016 >1 × 10⁸   >1 × 10⁸   >1 × 10⁸   1 × 10⁵ E. coli 10121 CTX-M-2 0.19 0.016 0.016 >1 × 10⁸   >1 × 10⁸   >1 × 10⁸   3 × 10⁵ E. coli SYD CMY-2 0.12 0.012 0.012 >1 × 10⁸   >1 × 10⁸   >1 × 10⁸   3 × 10³ E. coli MAR^(e) Overexpressed AmpC 16 >32 2 1 × 10² >1 × 10⁸   >1 × 10⁸   6 × 10² E. coli HB4^(d) (OmpC-, OmpF-) 0.12 1 0.25 1 × 10¹ >1 × 10⁸   1 × 10⁴ 6 × 10² E. aerogenes 1085 TEM-24 0.12 0.19 0.023 >1 × 10⁸   >1 × 10⁸   >1 × 10⁸   2 × 10¹ E. cloacae 5434 (wild type) 0.38 0.016 0.032 >1 × 10⁸   1 × 10⁶ 1 × 10² 1 × 10¹ E. cloacae CVB VEB-1 0.12 0.12 0.12 1 × 10⁴ >1 × 10⁸   1 × 10⁵ 6 × 10¹ E. cloacae CON^(e) Overexpressed AmpC 0.12 1 0.12 1 × 10⁷ >1 × 10⁸   >1 × 10⁸   6 × 10¹ E. cloacae BLA^(e) Overexpressed AmpC 0.12 1 0.12 1 × 10⁷ >1 × 10⁸   1 × 10⁷ 1 × 10² C. freundii 7767 (wild type) 0.25 0.008 0.016 >1 × 10⁸   >1 × 10⁸   >1 × 10⁸   1 × 10² C. freundii 10107 TEM-1 + SHV-12 0.38 0.016 0.023 >1 × 10⁸   >1 × 10⁸   >1 × 10⁸   2 × 10² C. freundii 10135 CTX-M-15 0.38 0.016 0.023 >1 × 10⁸   >1 × 10⁸   >1 × 10⁸   3 × 10¹ C. freundii MAU^(e) Overexpressed AmpC + TEM-3 1 8 1 1 × 10⁵ 5 × 10² 3 × 10¹ 1 × 10² S. typhimurium 1081 CTX-M-1 0.25 0.19 0.032 >1 × 10⁸   >1 × 10⁸   >1 × 10⁸   4 × 10¹

TABLE 7 Sensitivity and specificity of SUPERCARBA, and modified SUPERCARBA with doripenem 0.012, 0.024, or 0.048 μg/ml replacing ertapenem 0.25 μg/ml.^(a) Detection medium ETP DORI DORI DORI Cloxa 0.048 0.024 0.012 Zinc Cloxa Cloxa Cloxa (SUPERCARBA) Zinc Zinc Zinc SN (%)^(a) 94.7 73.7 94.7 94.7 SN class D 100 76.5 100 94.1 SP (%) 76.2 80.9 76.2 14.3 ^(a)Abbreviations: SN, sensitivity; SP, specificity. ^(b) Sensitivity was determined for each Ambler class of carbapenemase.

TABLE 8 Limit of detection of SUPERCARBA medium for 60 carbapenemase- and/or ESBL/AmpC-producing enterobacterial isolates as compared to those obtained with modified-SUPERCARBA, i.e. with oxacillin replacing cloxacillin. MIC values of imipenem, ertapenem and meropenem are provided for each strain. Lowest detection limit (CFU/ml) ETP MIC (μg/ml) Cloxa Zinc ETP Strains -Lactamase content IPM^(a) ETP MEM (SUPERCARBA) Oxa Zinc Ambler class A carbapenemases (KPC) K. pneumoniae MUS KPC-2 + TEM-1 + SHV-11 + 0.75 4 1.5 1 × 10¹ 2 × 10¹ SHV-12 E. cloacae HPTU KPC-2 + TEM-1 + SHV-11 2 4 1.5 1 × 10¹ 1 × 10¹ Ambler class B carbapenemases K. pneumoniae NDM-1 + OXA-1 1.5 6 2 1 × 10¹ 1 × 10¹ OMA419 K. pneumoniae OM2 NDM-1 + TEM-1 + CTX-M-3 + 0.75 8 1.5 1 × 10¹ 1 × 10¹ SHV-11 + OXA-1 P. stuartii PS1 NDM-1 + CMY-6 + OXA-1 12 0.38 1.5 1 × 10⁷ >5 × 10⁷   E. coli JAP IMP-1 0.5 3 0.5 1 × 10⁴ 4 × 10⁴ E. cloacae TAW IMP-8 + SHV-12 0.75 0.5 0.5 1 × 10² 2 × 10¹ Ambler class D carbapenemases K. pneumoniae BEL OXA-48 1 4 1 1 × 10¹ 1 × 10¹ K. pneumoniae SCO OXA-48 0.5 0.75 0.25 1 × 10¹ 1 × 10¹ K. pneumoniae ROU OXA-48 + CTX-M-15 0.5 1.5 0.25 1 × 10¹ 2 × 10¹ K. pneumoniae BAJ OXA-48 + TEM-1 + CTX-M-15 + 0.5 1.5 0.38 1 × 10¹ 1 × 10¹ SHV-28 K. pneumoniae BEN OXA-48 + TEM-1 + CTX-M-15 + 0.38 1 0.25 1 × 10¹ 1 × 10¹ SHV-28 K. pneumoniae SIC OXA-48 + CTX-M-15 + SHV-28 0.25 1 0.25 1 × 10¹ 1 × 10¹ K. pneumoniae AMS OXA-48 + TEM-1 + CTX-M-15 + 0.5 2 0.38 1 × 10¹ 2 × 10¹ OXA-1 K. pneumoniae ELK OXA-48 + TEM-1 + CTX-M-15 + 0.5 3 0.38 1 × 10¹ 1 × 10¹ SHV-11 K. pneumoniae VSG OXA-48 + TEM-1 + CTX-M-15 + 0.75 3 0.75 1 × 10¹ 1 × 10¹ OXA-1 E. coli ROB OXA-48 0.5 0.75 0.25 2 × 10¹ 6 × 10¹ E. coli BOU OXA-48 + CTX-M-15 0.5 0.75 0.125 2 × 10¹ 1 × 10¹ E. coli AME OXA-48 + CTX-M-24 0.25 0.5 0.19 2 × 10¹ 1 × 10¹ E. coli BON OXA-48 + TEM-1 + CTX-M-24 0.38 0.5 0.19 1 × 10¹ 2 × 10¹ E. cloacae TUR OXA-48 + SHV-5 0.5 0.5 0.5 1 × 10¹ 1 × 10¹ C. koseri VER OXA-48 0.75 2 0.38 1 × 10¹ 2 × 10¹ K. pneumoniae HOL OXA-181 + CTX-M-15 1 4 1 1 × 10¹ 1 × 10² K. pneumoniae OMA OXA-181 + CTXM-15 + OXA-1 0.5 2 0.5 1 × 10¹ 3 × 10¹ P. rettgeri RAP OXA-181 + OXA-1 8 1 2 5 × 10² 5 × 10¹ Non-carbapenemase producers K. pneumoniae 7725 SHV-1 0.19 0.006 0.032 >1 × 10⁸   >1 × 10⁸   K. pneumoniae 648236^(d) SHV-2a 0.25 2 0.38 1 × 10² 3 × 10¹ K. pneumoniae BER^(d) SHV-28 + TEM-1 1 4 1 1 × 10² 1 × 10² K. pneumoniae 1025 CTX-M-14 + TEM-1 + SHV-11 0.12 0.016 0.016 >1 × 10⁸   >1 × 10⁸   K. pneumoniae BED^(d) CTX-M-15 + TEM-1 + SHV-11 1.5 >32 4 1 × 10¹ 4 × 10¹ K. pneumoniae ALE^(d) CTX-M-15 + SHV-1 1 >32 4 1 × 10⁵ 1 × 10⁵ K. pneumoniae KDH^(e) DHA-2 0.12 0.5 0.12 1 × 10² 1 × 10¹ E. coli 6367 (wild type) 0.19 0.006 0.012 >1 × 10⁸   >1 × 10⁸   E. coli 1082 TEM-1 0.19 0.019 0.016 >1 × 10⁸   >1 × 10⁸   E. coli 1034 TEM-1 + SHV-38 0.19 0.006 0.016 >1 × 10⁸   >1 × 10⁸   E. coli 1048 TEM-1 + SHV-2a 0.19 0.012 0.016 >1 × 10⁸   >1 × 10⁸   E. coli 1008 TEM-1 + CTX-M-1 0.19 0.016 0.016 >1 × 10⁸   >1 × 10⁸   E. coli 10121 CTX-M-2 0.19 0.016 0.016 >1 × 10⁸   1 × 10¹ E. coli FOR CTX-M-15 0.12 0.012 0.012 >1 × 10⁸   1 × 10¹ E. coli EVB VEB-1 0.12 0.012 0.012 >1 × 10⁸   1 × 10¹ E. coli 1092 OXA-1 0.12 0.19 0.023 >1 × 10⁸   1 × 10¹ E. coli ECA ACC-1 0.12 0.012 0.012 >1 × 10⁸   5 × 10³ E. coli SYD CMY-2 0.12 0.012 0.012 >1 × 10⁸   1 × 10¹ E. coli MET Chromosome-encoded extended- 0.12 0.012 0.012 >1 × 10⁸   1 × 10¹ spectrum cephalosporinase E. coli MAR^(e) Overexpressed AmpC 16 >32 2 1 × 10² 1 × 10¹ E. coli HB4^(d) (OmpC-, OmpF-) 0.12 1 0.25 1 × 10¹ >1 × 10⁸   E. aerogenes 1085 TEM-24 0.12 0.19 0.023 >1 × 10⁸   1 × 10¹ E. cloacae 5434 (wild type) 0.38 0.016 0.032 >1 × 10⁸   >1 × 10⁸   E. cloacae CLO CTX-M-15 0.12 0.12 0.12 1 × 10⁷ 1 × 10¹ E. cloacae 10111^(e) TEM-1 + CTX-M-15 0.5 0.75 0.094 >1 × 10⁸   1 × 10¹ E. cloacae CVB VEB-1 0.12 0.12 0.12 1 × 10⁴ 1 × 10¹ E. cloacae ARF^(e) Overexpressed AmpC 0.12 1 0.12 1 × 10⁷ 1 × 10¹ E. cloacae BLA^(e) Overexpressed AmpC 0.12 1 0.12 1 × 10⁷ 1 × 10¹ C. freundii 7767 (wild type) 0.25 0.008 0.016 >1 × 10⁸   >1 × 10⁸   C. freundii 10107 TEM-1 + SHV-12 0.38 0.016 0.023 >1 × 10⁸   1 × 10¹ C. freundii 10135 CTX-M-15 0.38 0.016 0.023 >1 × 10⁸   1 × 10¹ C. freundii MAU^(e) Overexpressed AmpC + TEM-3 1 8 1 1 × 10⁵ 1 × 10¹ S. typhimurium 1081 CTX-M-1 0.25 0.19 0.032 >1 × 10⁸   1 × 10¹ P. mirabilis 1031 CTX-M-14 + TEM-1 + SHV-11 1.5 0.047 0.032 >1 × 10⁸   1 × 10¹ P. mirabilis PMA ACC-1 0.25 0.094 0.064 >1 × 10⁸   >1 × 10⁸  

TABLE 9 Sensitivity and specificity of SUPERCARBA, ETP-Oxa-Zinc.^(a) Detection medium ETP Cloxa Zinc ETP (SUPERCARBA) Oxa -Zinc SN (%)^(a) 88 88 SN class A^(b) 100 100 SN class B 40 40 SN class D 100 100 SP (%) 82.8 80 ^(a)Abbreviations: SN, sensitivity; SP, specificity. ^(b)Sensitivity was determined for each Ambler class of carbapenemase.

TABLE 10 Limit of detection of SUPERCARBA medium (Drigalski + ETP + Cloxa + Zinc) for 51 carbapenemase- and/or ESBL/AmpC-producing enterobacterial isolates as compared to those obtained with modified-SUPERCARBA that contained Trypticase-soy agar replacing Drigalski agar supplemented with vancomycin (20 μg/ml) and ETP + Cloxa + Zinc. MIC values of imipenem, ertapenem and meropenem are provided for each strain. Lowest detection limit (CFU/ml) ETP- ETP Cloxa Zinc- MIC (μg/ml) Cloxa Zinc Vanco-modified Strains -Lactamase content IPM^(a) ETP MEM (SUPERCARBA) SUPERCARBA Ambler class A carbapenemases (KPC) K. pneumoniae MUS KPC-2 + TEM-1 + SHV-11 + 0.75 4 1.5 1 × 10¹ 1 × 10¹ SHV-12 E. cloacae HPTU KPC-2 + TEM-1 + SHV-11 2 4 1.5 1 × 10¹ 1 × 10¹ Ambler class B carbapenemases K. pneumoniae NDM-1 + OXA-1 1.5 6 2 1 × 10¹ 1 × 10¹ OMA419 K. pneumoniae OM2 NDM-1 + TEM-1 + CTX-M-3 + 0.75 8 1.5 1 × 10¹ 1 × 10¹ SHV-11 + OXA-1 P. stuartii PS1 NDM-1 + CMY-6 + OXA-1 12 0.38 1.5 1 × 10⁷ 1 × 10² E. coli JAP IMP-1 0.5 3 0.5 1 × 10⁴ 3 × 10¹ E. cloacae TAW IMP-8 + SHV-12 0.75 0.5 0.5 1 × 10² 2 × 10² Ambler class D carbapenemases K. pneumoniae BEL OXA-48 1 4 1 1 × 10¹ 1 × 10¹ K. pneumoniae SCO OXA-48 0.5 0.75 0.25 1 × 10¹ 1 × 10¹ K. pneumoniae ROU OXA-48 + CTX-M-15 0.5 1.5 0.25 1 × 10¹ 1 × 10¹ K. pneumoniae BAJ OXA-48 + TEM-1 + CTX-M-15 + 0.5 1.5 0.38 1 × 10¹ 1 × 10¹ SHV-28 K. pneumoniae BEN OXA-48 + TEM-1 + CTX-M-15 + 0.38 1 0.25 1 × 10¹ 1 × 10¹ SHV-28 K. pneumoniae SIC OXA-48 + CTX-M-15 + SHV-28 0.25 1 0.25 1 × 10¹ 1 × 10¹ K. pneumoniae AMS OXA-48 + TEM-1 + CTX-M-15 + 0.5 2 0.38 1 × 10¹ 1 × 10¹ OXA-1 K. pneumoniae ELK OXA-48 + TEM-1 + CTX-M-15 + 0.5 3 0.38 1 × 10¹ 1 × 10¹ SHV-11 K. pneumoniae VSG OXA-48 + TEM-1 + CTX-M-15 + 0.75 3 0.75 1 × 10¹ 1 × 10¹ OXA-1 E. coli ROB OXA-48 0.5 0.75 0.25 2 × 10¹ 1 × 10¹ E. coli BOU OXA-48 + CTX-M-15 0.5 0.75 0.125 2 × 10¹ 1 × 10¹ E. coli AME OXA-48 + CTX-M-24 0.25 0.5 0.19 2 × 10¹ 1 × 10¹ E. coli BON OXA-48 + TEM-1 + CTX-M-24 0.38 0.5 0.19 1 × 10¹ 1 × 10¹ E. cloacae TUR OXA-48 + SHV-5 0.5 0.5 0.5 1 × 10¹ 1 × 10¹ C. koseri VER OXA-48 0.75 2 0.38 1 × 10¹ 1 × 10¹ K. pneumoniae HOL OXA-181 + CTX-M-15 1 4 1 1 × 10¹ 1 × 10² K. pneumoniae OMA OXA-181 + CTXM-15 + OXA-1 0.5 2 0.5 1 × 10¹ 1 × 10¹ P. rettgeri RAP OXA-181 + OXA-1 8 1 2 5 × 10² 1 × 10¹ Non-carbapenemase producers K. pneumoniae 7725 SHV-1 0.19 0.006 0.032 >1 × 10⁸   >1 × 10⁸   K. pneumoniae 648236^(d) SHV-2a 0.25 2 0.38 1 × 10² 3 × 10¹ K. pneumoniae BER^(d) SHV-28 + TEM-1 1 4 1 1 × 10² 1 × 10¹ K. pneumoniae BED^(d) CTX-M-15 + TEM-1 + SHV-11 1.5 >32 4 1 × 10¹ 1 × 10¹ K. pneumoniae ALE^(d) CTX-M-15 + SHV-1 1 >32 4 1 × 10⁵ 1 × 10¹ E. coli 1034 TEM-1 + SHV-38 0.19 0.006 0.016 >1 × 10⁸   >1 × 10⁸   E. coli 1048 TEM-1 + SHV-2a 0.19 0.012 0.016 >1 × 10⁸   >1 × 10⁸   E. coli 1008 TEM-1 + CTX-M-1 0.19 0.016 0.016 >1 × 10⁸   >1 × 10⁸   E. coli 10121 CTX-M-2 0.19 0.016 0.016 >1 × 10⁸   >1 × 10⁸   E. coli FOR CTX-M-15 0.12 0.012 0.012 >1 × 10⁸   >1 × 10⁸   E. coli EVB VEB-1 0.12 0.012 0.012 >1 × 10⁸   >1 × 10⁸   E. coli ECA ACC-1 0.12 0.012 0.012 >1 × 10⁸   >1 × 10⁸   E. coli SYD CMY-2 0.12 0.012 0.012 >1 × 10⁸   >1 × 10⁸   E. coli MET Chromosome-encoded extended- 0.12 0.012 0.012 >1 × 10⁸   >1 × 10⁸   spectrum cephalosporinase E. coli MAR^(e) Overexpressed AmpC 16 >32 2 1 × 10² >1 × 10⁸   E. coli HB4^(d) (OmpC-, OmpF-) 0.12 1 0.25 1 × 10¹ 1 × 10¹ E. aerogenes 1085 TEM-24 0.12 0.19 0.023 >1 × 10⁸   >1 × 10⁸   E. cloacae CLO CTX-M-15 0.12 0.12 0.12 1 × 10⁷ >1 × 10⁸   E. cloacae 10111^(e) TEM-1 + CTX-M-15 0.5 0.75 0.094 >1 × 10⁸   2 × 10⁷ E. cloacae CVB VEB-1 0.12 0.12 0.12 1 × 10⁴ >1 × 10⁸   E. cloacae CON^(e) Overexpressed AmpC 0.12 1 0.12 1 × 10⁷ 1 × 10⁷ E. cloacae BLA^(e) Overexpressed AmpC 0.12 1 0.12 1 × 10⁷ 2 × 10⁷ C. freundii 10107 TEM-1 + SHV-12 0.38 0.016 0.023 >1 × 10⁸   >1 × 10⁸   C. freundii 10135 CTX-M-15 0.38 0.016 0.023 >1 × 10⁸   >1 × 10⁸   C. freundii MAU^(e) Overexpressed AmpC + TEM-3 1 8 1 1 × 10⁵ 1 × 10¹ S. typhimurium 1081 CTX-M-1 0.25 0.19 0.032 >1 × 10⁸   >1 × 10⁸  

TABLE 11 Sensitivity and specificity of SUPERCARBA and modified SUPERCARBA screening media. Detection medium ETP Cloxa Zinc Supercarba (SUPERCARBA) modified SN (%)^(a) 92 100 SP (%) 80.8 77 ^(a)Abbreviations: SN, sensitivity; SP, specificity.

REFERENCES

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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The invention claimed is:
 1. A method for detecting carbapenem-resistant bacteria in a test sample comprising the following successive steps: a) inoculating a culture medium with said test sample, wherein said culture medium comprises a carbapenem, a M-type penicillin and a carbapenemase activator selected from the group consisting of divalent cations and mixtures thereof, and is suitable for growth of class A, B and D carbapenemase-producing bacteria; and b) incubating said culture medium under conditions suitable for growth of carbapenem-resistant bacteria; and c) detecting colonies formed on said culture medium, wherein detection of colonies is an indication of the presence in said test sample of at least one species of carbapenem-resistant bacteria producing a carbapenemase belonging to class A, B or D.
 2. The method according to claim 1, wherein the carbapenem is selected from the group consisting of ertapenem, biapenem, doripenem, imipenem, meropenem, tebipenem, panipenem and mixtures thereof.
 3. The method according to claim 1, wherein the carbapenem is ertapenem and the concentration of ertapenem in the culture medium is from 0.1 to 1 μg/ml.
 4. The method according to claim 1, wherein the carbapenem is doripenem and the concentration of doripenem in the culture medium is from 0.01 to 0.1 μg/ml.
 5. The method according to claim 1, wherein the carbapenemase activator is a divalent cation selected from the group consisting of zinc, magnesium, manganese and mixtures thereof.
 6. The method according to claim 1, wherein the divalent cation is zinc.
 7. The method according to claim 1, wherein the M-type penicillin is selected from the group consisting of cloxacillin, dicloxacillin, flucloxacillin, oxacillin, methicillin, nafcillin and mixtures thereof.
 8. The method according to claim 1, wherein the M-type penicillin is cloxacillin.
 9. The method according claim 1, wherein the carbapenem is ertapenem, the divalent cation is zinc, and the M-type penicillin is cloxacillin. 